Project Summary Background: Triple-negative (estrogen receptor [ER]/progesterone receptor [PR]/human epidermal growth factor receptor 2 [HER2]-negative) breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Approximately 34% of patients with TNBC will experience distant recurrence within 3 years and eventually develop chemotherapy-resistant, incurable disease. Therefore, development of novel treatments is necessary. Accumulating data demonstrate that microRNA (miR)-146a, which is part of the miR-146 family, inhibits cancer cell proliferation, invasion, and metastasis in human cancers, including TNBC, suggesting that miR-146a functions as a tumor suppressor in tumor cells. Previously, our group demonstrated that miR-146a negatively regulates NF-?B activation to inhibit tumor growth in TNBC. Furthermore, our data suggested that miR-146a suppresses TNBC colonization through the cellular transfer of miR-146a by exosomes at distant sites and the delivery of miR-146a mimic influences the cells at metastatic sites in vivo. Thus, miR-146a may have potential to block TNBC cell colonization at distant sites. We now seek to test the therapeutic value of miR-146a in the prevention or treatment of TNBC metastasis. Hypothesis: We hypothesize that miR-146a effectively blocks the tumor colonization of disseminated TNBC cells at metastatic sites. This hypothesis will be tested in two specific aims: 1) To evaluate the therapeutic value of miR-146a in blocking tumor colonization at metastatic sites, and 2) To determine the mechanism of miR-146a- mediated suppression of tumor metastasis. Approach: First, to address the impact of miR-146a on preventing human TNBC cell colonization, we will intravenously inject scramble miR or miR-146a mimic nanoparticles once circulating tumor cells are detected in xenograft mouse models. Next, to evaluate the efficacy of miR-146a in inhibiting tumor metastasis in a clinically relevant model, we will intravenously inject scramble miR or miR-146a mimic nanoparticles into TNBC patient- derived xenograft mouse models and determine if miR-146a blocks TNBC metastasis to distant organs. Finally, to identify which cells are influenced by miR-146a and which miR-146a-regulated pathways are involved in tumor metastasis, we will use our innovative transgenic mouse model that expresses a miR-146a-targeted GFP reporter to monitor miR-146a activity in specific cell types during tumor metastasis. Innovation and Significance: TNBC remains a highly aggressive and fatal subtype of breast cancer, with the highest mortality in young minority women. Developing effective therapies to treat this disease is crucial. The data from this study will not only provide a novel therapeutic approach to block tumor metastasis in TNBC, but will also identify a new molecular mechanism responsible for tumor metastasis. Since our preliminary studies support the suppressive role of miR-146a in tumor metastasis, the tight focus of this project will allow us to establish the mechanism-driven innovative pre-clinical studies that could lead to a first-in-human clinical trial.